Integrated sample processing system

ABSTRACT

An integrated sample purification system includes a housing, a sample container rack, a filter holder, and a cylindrical magnet. The sample container rack and the filter device holder are disposed in the housing. The sample container rack is configured to hold one or more sample containers, the filter device holder is configured to hold one or more filter devices. The cylindrical magnet is adjacent to and external to the sample container rack, and is rotated about a central, longitudinal axis of the magnet by an electric motor disposed in the housing to lyse cells. Molecules of interest in the lysed cells are purified using filters that bind specifically to the molecules of interest. The system is readily amenable to automation and rapid purification and analysis of molecules of interest, such as nucleic acids and proteins.

The instant application is a continuation application of U.S.application Ser. No. 16/158,129, filed on Oct. 11, 2018, which is acontinuation of U.S. application Ser. No. 14/955,681, filed Dec. 1,2015, now U.S. Pat. No. 10,125,388 which is a continuation-in-partapplication of U.S. application Ser. No. 13/765,399, filed on Feb. 12,2013, now U.S. Pat. No. 9,217,174, which is a continuation applicationof U.S. application Ser. No. 12/886,144, filed on Sep. 20, 2010, nowU.S. Pat. No. 8,399,190, which claims the priority of U.S. ProvisionalApplication No. 61/272,396, filed on Sep. 21, 2009.

U.S. application Ser. No. 14/955,681, now U.S. Pat. No. 10,125,388, isalso a continuation-in-part application of U.S. application Ser. No.14/294,683, filed on Jun. 3, 2014, now U.S. Pat. No. 9,493,815, which isa continuation of U.S. application Ser. No. 13/446,291, filed on Apr.13, 2012, now U.S. Pat. No. 8,828,912, which is a continuation-in-partof U.S. application Ser. No. 12/886,201, filed on Sep. 20, 2010, nowU.S. Pat. No. 8,623,789, which claims priority to U.S. ProvisionalApplication No. 61/272,397, filed on Sep. 21, 2009, and U.S. applicationSer. No. 13/446,291, filed Apr. 13, 2012, also claims priority to U.S.Provisional Application No. 61/475,107, filed on Apr. 13, 2011.

U.S. application Ser. No. 14/955,681, now U.S. Pat. No. 10,125,388, isalso a continuation-in-part of U.S. application Ser. No. 14/011,267,filed on Aug. 27.2013, now a U.S. Pat. No. 9,428,746, which is acontinuation-in-part application of U.S. application Ser. No.13/682,551, filed on Nov. 20, 2012, now U.S. Pat. No. 8,574,923, whichis a divisional application of U.S. application Ser. No. 12/213,942,filed on Jun. 26, 2008, now abandoned, which is a continuation-in-partapplication of U.S. application Ser. No. 11/933,113, filed on Oct. 31,2007, now U.S. Pat. No. 7,759,112. U.S. application Ser. No. 14/011,267also claims priority of U.S. Provisional Application No. 61/697,116,filed on Sep. 5, 2012 and U.S. Provisional Application No. 61/693,963,filed on Aug. 28, 2012.

U.S. application Ser. No. 14/955,681, now U.S. Pat. No. 10,125,388, isalso a continuation-in-part application of U.S. application Ser. No.13/314,734, filed on Dec. 8, 2011, now U.S. Pat. No. 10,300,482, whichclaims the priority of U.S. Provisional Application No. 61/421,414,filed on Dec. 9, 2010.

The entirety of all of the aforementioned applications is incorporatedherein by reference.

FIELD

The present invention relates generally to an integrated sampleprocessing system for isolating and/or purifying molecules of interest,such as nucleic acids and proteins, especially from difficult samplematrices and/or difficult-to-disrupt organisms, as well as methodsamenable to automation for isolating and/or purifying nucleic acids froma sample using magnetically-induced vortexing in combination with solidmonolith filters.

BACKGROUND

Molecular testing is emerging as a gold standard for some diagnostictests due to their speed, sensitivity and specificity. LaboratoryDeveloped Tests (LDTs) “are now one of the fastest growing segments inthe in vitro diagnostic (IVD) market. Sample preparation is critical tothe validity of the test, but frequently presents a bottleneck forclinical molecular biology workflows and diagnostic tests. While thereare many molecular detection modalities, there are only a handful ofautomated sample preparation workflow strategies. Existing instrumentsthat are built around these sample preparation strategies andchemistries range in cost from $17-$150 k, and yet they still do notprovide an integrated method for processing difficult sample matricessuch as raw sputum and/or difficult-to-disrupt organisms such asgram-positive bacteria and the acid fast bacilli (i.e., Mycobacterium).

Acid fast bacilli, including Mycobacterium strains are typicallyisolated from sputum of infected patients. They are known as “acid-fastbacilli” because of their lipid-rich cell envelope, which is relativelyimpermeable to various basic dyes unless the dyes are combined withphenol. Sputum is thick, viscous and difficult to process. Most sputumspecimens for analysis contain various amounts of organic debris and avariety of contaminating, normal, or transient bacterial flora. Chemicaldecontamination/processing is typically used to reduce the viscosity andkill the contaminants while allowing recovery of the mycobacteria.Because of their unique cell envelope containing mycolic acids and ahigh lipid content, however, the cells are hydrophobic and tend to clumptogether. This renders them impermeable to the usual stains, such as theGram stain. Two types of acid-fast stains are generally used, carbolfuchsin and fluorochromes, such as auramine or auramine-rhodamine. Oncestained, the cells resist decolorization with acidified organicsolvents, and are therefore called “acid-fast”. However, they retainfuchsine or auramine staining after successive or simultaneous treatmentwith acid and alcohol.

The sensitivity of acid-fast smear microscopy for Mycobacterium speciesis poor. The sensitivity of microscopy is influenced by numerousfactors, such as the prevalence and severity of disease, the type ofspecimen, the quality of specimen collection, the number ofMycobacterium cells present in the specimen, the method of processing(direct or concentrated), the method of centrifugation, the stainingtechnique, and the quality of the examination. It is recommended that anegative result should only be reported following the examination of atleast 100 (in low-income countries) and preferably 300 (inindustrialized countries) microscopic immersion view fields (orequivalent fluorescent view fields). Therefore, when microscopy isperformed correctly, it can be time-consuming and laborious.

Mycobacterium strains are slow-growing bacilli, with a usual generationtime of 12 to 18 hours. Colonies usually become visible only after a1-week to 8-week incubation time. Samples which contain a lowconcentration of Mycobacterium cells further necessitate severalsubcultures. Mycobacterium cultures on specific media can allow for theidentification of the particular Mycobacterium species contained in thebiological sample. However, this is time-consuming, especially for thosepatients who are only at the beginning of the infection process.

Nucleic acid hybridization tests have been developed to detect strainsof Mycobacterium in a biological sample. The first tests utilized directprobe hybridization. However, the concentration in Mycobacterium cellscontained in a sample collected from a patient is usually too low togive a positive hybridization signal. Tests utilizing PCR amplificationhave therefore been developed. For example, the Gen-Probe® kitcommercialized as “Amplified™ Mycobacterium tuberculosis direct test”kit, or MTD test kit, (Gen-Probe Inc., San Diego, Calif. 92121, USA)utilizes the amplification of MtbC-specific rRNA (Transcription-MediatedAmplification), followed by amplicon detection in accordance with theGen-Probe HPA method (Hybridization Protection Assay).

In view of the above-described limitations, there is a need for simpleand efficient systems integrating sample homogenization, lysis ofdifficult-to-disrupt microorganisms, and polynucleotide purification tomeet the needs of both clinical laboratories and users alike.

SUMMARY

In one aspect, the present application provides an integrated samplepurification system comprising housing, a sample container rack, afilter tip rack, and a cylindrical magnet. The sample container rack andthe filter tip rack are disposed in the housing. The sample containerrack is configured to hold one or more sample containers, the filter tiprack is configured to hold one or more filter tips. The cylindricalmagnet is adjacent to and external to the sample container rack, and isrotatably driven about a central, longitudinal axis of the magnet by anelectric motor disposed in the housing.

In some embodiments, the housing comprises one or more reagent rackscontaining one or more reagents.

In some embodiments, the system comprises a plurality of the samplecontainers, a plurality of the filter tips and one or more reagentracks.

In certain embodiments, the cylindrical magnet has magnetic polessymmetrically disposed along and around the longitudinal axis of themagnet. In other embodiments, the cylindrical magnet has opposingmagnetic poles disposed at opposite longitudinal ends of the magnet. Inyet other embodiments, the cylindrical magnet is an electromagnet.

In one embodiment, the one or more sample containers are sealed andconfigured to maintain a closed system following introduction of one ormore reagent solutions.

In one embodiment, the system further comprises a reagent rack disposedin the housing, whereby the reagent rack comprises reagents stored inseparate, sealed wells within the rack.

When in use, the sample container includes a magnetic stirrer and aplurality of beads configured so that when the sample containercomprises cellular material and the cylindrical magnet is rotated aboutits longitudinal axis, the magnetic stirrer spins and agitates the beadsto undergo chaotic mixing of the cellular material, resulting in samplehomogenization and cell disruption.

In one embodiment, the beads comprise glass, plastic, ceramic material,minerals, metal or a combination thereof. In a particular embodiment,the beads are silica beads.

In one embodiment, the beads have diameters within the range of 10-1000μm.

In one embodiment, the magnetic stirrer comprises a metal or an alloy.In a particular embodiment, the magnetic stirrer comprises stainlesssteel. In another embodiment, the magnetic stirrer comprises an alloycore coated with a polymer. In a particular embodiment, the magneticstirrer comprises an alloy core coated with a polymer, whereby the alloycore comprises neodymium iron boron or samarium cobalt and/or where thepolymer is PTFE or parylene.

In another aspect, an automated nucleic acid purification systemincludes the above-described features in combination with an automatedpipetting system and one or more robotic arms configured toautomatically dispense reagents into the one or more sample containersand dispose of sample materials and reagents in a predetermined manner.When in use, the automated purification system includes a plurality ofthe sample containers, each containing a stirrer and beads, a pluralityof the filter tips and one or more reagent racks.

In another aspect, a method for purifying target molecules from asample, includes the steps of (a) providing a sample purification systemin accordance with the present disclosure; (b) placing a sample with amagnetic stirrer and a plurality of beads in a sample container; (c)placing the sample container on the sample container rack, (d) rotatingthe cylindrical magnet about its longitudinal axis so that the magneticstirrer spins and agitates the beads to a degree sufficient tohomogenize the sample and disrupt the cells in the sample to form a celllysate; (e) flowing at least a portion of the cell lysate through afirst opening of a filter tip so that target molecules in the celllysate bind to the filter in the filter tip; (f) expelling an unboundportion of the cell lysate from the filter tip via the first opening,where the unbound portion passes through the filter at least two timesbefore exiting the filter tip; and (g) eluting the target moleculesbound to the filter by flowing an elution buffer in through the firstopening of the filter tip and expelling the elution buffer from thefilter tip via the first opening, wherein the elution buffer passesthrough the filter at least two times before exiting the filter tip.

In some embodiments, the target molecules are polynucleotide molecules.In one embodiment, the sample comprises sputum. In a particularembodiment, the sputum sample is suspected of containing MycobacteriumTuberculosis (MTB) and the method further includes the step ofamplifying the eluted polynucleotide molecules with primers specific forMTB and determining whether the polynucleotide molecules comprise MTBDNA.

In another embodiment, the method for purifying target moleculescomprises the use of an automated purification system further comprisingan automated pipetting system and one or more robotic arms configured toautomatically dispense reagents into the one or more sample containersand dispose of sample materials and reagents in a predetermined manner.In this case, each of the above-described steps are repeated in each ofa plurality of sample containers using an equivalent number of filtertips in combination with one or more reagent racks.

BRIEF DESCRIPTION OF DRAWINGS

The detailed description will refer to the following drawings in which:

FIG. 1 is a flow chart showing an embodiment of an integrated method forlysing cells and purifying nucleic acids therefrom.

FIG. 2 depicts an exemplary single-channel nucleic acid purificationsystem according to one embodiment.

FIG. 3 shows exemplary positions for placement of the magnet relative toa sample lysis chamber.

FIG. 4 depicts an exemplary pipette filter tip.

FIGS. 5A and 5B are schematic illustrations depicting an automated8-channel nucleic acid purification system according to anotherembodiment.

FIG. 6 depicts a disposable transport device according to anotherembodiment.

FIG. 7 illustrates an exemplary sequence of steps for MagVor/filter tippurification of nucleic acids from sputum.

DETAILED DESCRIPTION

In describing preferred embodiments of the present invention, specificterminology is employed for the sake of clarity. However, the inventionis not intended to be limited to the specific terminology so selected.It is to be understood that each specific element includes all technicalequivalents which operate in a similar manner to accomplish a similarpurpose.

FIG. 1 is a flow chart depicting exemplary process steps in anintegrated method for lysing cells and purifying molecules of interest,such as nucleic acids or proteins therefrom. The method 10 includesplacing a sample tube containing a liquid sample suspension, a magneticstirrer and cell lysis beads on a sample rack in the proximity of amagnet (step 11); homogenizing the sample suspension by rotating themagnet at a speed sufficient to lyse cells in the sample suspension inthe presence of the magnetic stirrer and cell lysis beads (step 13);flowing the homogenized sample suspension through a filter matrix underconditions that the molecules of interest bind to the filter matrix(step 15); washing the filter matrix (step 17) and eluting boundmolecules of interest from the filter matrix (step 19). In someembodiments, the sample tube is pre-packed with a magnetic stirrer,and/or cell lysis beads, and/or reagents that facilitate cell lysisand/or preserve the integrity of the target molecules.

The liquid sample suspension is a sample suspended in liquid lysismedium. Exemplary samples may include biological samples, environmentalsamples or non-nature samples. Exemplary biological samples may includetissue samples, biological fluid samples, cell samples, fungal samples,protozoan samples, bacterial samples, and virus samples. Tissue samplesinclude tissues isolated from any animal or plant. Biological samplesinclude, but are not limited to, blood, cord blood, plasma, buffy coat,urine, saliva, sputum, NALC-treated sputum, nasopharyngeal swabs (NPS),nasopharyngeal aspirates (NPA), gastric aspirate, concentrated coughcollection, cerebrospinal fluid, buccal, lavages (e.g. bronchial),pleural fluids, stool, and leukophoresis samples. Cell samples furtherinclude cultured cells, fresh or frozen cells and tissues from any cellsources, including fixed, paraffin-embedded (FFPE) tissues. Bacteriasamples include, but are not limited to, cultured bacteria, isolatedbacteria, and bacteria within any of the previously stated biologicalsamples. Virus samples include, but are not limited to, culturedviruses, isolated viruses, and viruses within any of the previouslystated biological samples. Environmental samples include, but are notlimited to, air samples, water samples, soil samples, rock samples andany other samples obtained from a natural environment. The artificialsamples include any sample that does not exist in a natural environment.Examples of “artificial samples” include, but are not limited to,purified or isolated materials, cultured materials, synthesizedmaterials and any other man-made materials.

The liquid lysis medium can be isotonic, hypotonic, or hypertonic. Insome embodiments, the liquid lysis medium is aqueous. In certainembodiments, the liquid lysis medium contains a buffer and/or at leastone salt or a combination of salts. In some embodiments, the pH of theliquid lysis medium ranges from about 5 to about 8, from about 6 to 8,or from about 6.5 to about 8.5. A variety of pH buffers may be used toachieve the desired pH. Suitable buffers include, but are not limitedto, Tris, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris propane, BES,MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, TEA, HEPPS,Tricine, Gly-Gly, Bicine, and a phosphate buffer (e.g., sodium phosphateor sodium-potassium phosphate, among others). The liquid lysis mediummay comprise from about 10 mM to about 100 mM buffer, about 25 mM toabout 75 mM buffer, or from about 40 mM to about 60 mM buffer, amongothers. The type and amount of the buffer used in the liquid medium canvary from application to application. In some embodiments, the liquidlysis medium has a pH of about 7.4, which can be achieved using about 50mM Tris buffer. In some embodiments the liquid lysis medium is water.

Eukaryotic cells, prokaryotic cells, and/or viruses may be suspended atany suitable concentration. Preferably, the samples contain cellssuspended in a liquid medium at a concentration that does not interferewith the movement of the magnetic stirrer. In some embodiments,eukaryotic cells and/or prokaryotic cells are suspended at aconcentration ranging from 1 to 1×10¹⁰ cells/ml, 1 to 1×10⁵ cells/ml, or1×10³ to 1×10⁴ cells/ml, among others. In some embodiments, virusparticles are suspended in a concentration ranging from 1 to 1×10¹³particles/ml, 1 to 1×10¹⁰ particles/ml, or 1×10⁵ to 1×10⁷ particles/ml.

In certain preferred embodiments, the sample is suspected of containingMTB. In one embodiment, the sample is a nasopharyngeal aspirate. Inanother embodiment, the sample is a nasopharyngeal swab.

As used herein, the term “cells” refers to eukaryotic cells, prokaryoticcells, viruses, endospores or any combination thereof Cells thus mayinclude bacteria, bacterial spores, fungi, virus particles,single-celled eukaryotic organisms (e.g., protozoans, yeast, etc.),isolated or aggregated cells from multi-cellular organisms (e.g.,primary cells, cultured cells, tissues, whole organisms, etc.), or anycombination thereof, among others.

The terms “sample” refer to any material that contains the targetmolecules or is suspected of containing the target molecules.

The term “nucleic acids” refers to individual nucleic acids andpolymeric chains of nucleic acids, including DNA and RNA, whethernaturally occurring or artificially synthesized (including analogsthereof), or modifications thereof, especially those modifications knownto occur in nature, having any length.

The term “sample container” refers to an elongated, generally tubularcontainer or vial for securing and/or processing a sample forpurification of nucleic acid or receiving reagents in combination with aprocessed sample. The sample containers need not be cylindrical and maybe slightly conical along their entire length or along a portionthereof.

The term “lyse” with respect to cells means disruption of the integrityof at least a fraction of the cells to release intracellular components,such as nucleic acids and proteins, from the disrupted cells.

The term “homogenize” means blending or vortexing (diverse elements e.g.stool, tissue, sputum, saliva) into a uniform mixture.

The terms “closed system” or “closed container” refers to tubes orcontainers that are sealed and operate in a substantially, if nottotally, closed manner to impede or prevent the introduction ofexogenous or external materials into (or out of) the tube or containerduring processing. Components of the closed systems or containers can bepre-sterilized prior to use at a manufacture site, sterilized at thepoint of use, and/or sterilized after a respective closed system isassembled and closed prior to use.

The term “single-use disposable” refers to a component that is notreused. That is, after completing its intended use, i.e., processing orproduction of a target sample or sample(s), it is disposed of.

As used herein, the terms “monolith adsorbent” or “monolithic adsorbentmaterial” refer to a porous, three-dimensional adsorbent material havinga continuous interconnected pore structure in a single piece, which maycomprise a rigid, self-supporting substantially monolithic structure. Amonolith is prepared, for example, by casting, sintering or polymerizingprecursors into a mold of a desired shape. The term “monolith adsorbent”or “monolithic adsorbent material” is meant to be distinguished from acollection of individual adsorbent particles packed into a bed formationor embedded into a porous matrix, in which the end product comprisesindividual adsorbent particles. Porous monolithic polymers are a newcategory of materials developed during the last decade. In contrast topolymers composed of very small beads, a monolith is a single,continuous piece of a polymer prepared using a simple molding process.The term “monolith adsorbent” or “monolithic adsorbent material” is alsomeant to be distinguished from a collection of adsorbent fibers orfibers coated with an adsorbent, such as filter papers or filter paperscoated with an adsorbent.

In one aspect, the present application provides an integrated samplepurification system comprising housing, a sample container rack, afilter tip rack, and a cylindrical magnet. The sample container rack andthe filter tip rack are disposed in the housing. The sample containerrack is configured to hold one or more sample containers, the filter tiprack is configured to hold one or more filter tips. The cylindricalmagnet is adjacent to and external to the sample container rack, and isrotatably driven about a central, longitudinal axis of the magnet by anelectric motor disposed in the housing.

FIG. 2 depicts an exemplary single-channel sample purification system100 according to one embodiment. The system 100 in FIG. 2 includeshousing 104, a sample container rack 108, a filter tip actuator/rack112, and a cylindrical magnet 116. The sample container rack 108 and thefilter tip rack 112 are disposed in the housing 104. A sample containerrack or stand 108 holds the sample container 120. The filter tipactuator/rack 112 in FIG. 2 is configured to hold a filter tip 124attached to syringe 176, which is configured so that a syringe plunger174 in the syringe 176 moves up and down to aspirate and dispense liquidthrough the filter tip 124. The plunger 174 is connected to an actuatorin the rack 112 that controls the plunger movement. The cylindricalmagnet 116 is adjacent to and external to the sample container rack 108,and is rotated about a central, longitudinal axis of the magnet 116 byan electric motor 130 disposed in the housing 104.

Each sample container 120 may contain one or more lysis chambers forlysing cells in a sample. Preferably, the sample container(s) 120 (andother system components) are sealed and configured to maintain a closedsystem before and after introduction of samples 144 and/or one or morereagent solutions. The container 120 may be sealed with a lid, cap, orcover. The sample container 120 can be made with any suitable material,size, and shape. In certain embodiments, the container 120 is made ofplastic. Preferably, the interior surface of the container 120 ischemically inert. The sample container 120 may, for example, be in theshape of a urine collection cup, a micro centrifuge tube (e.g., anEppendorf tube), a centrifuge tube, a vial, a microwell plate etc. Insome embodiments, the container 120 contains a singlecompartment/chamber for holding cells 180, beads 160, and a stirrer 156,as shown in FIG. 3 . In some embodiments, a given container 120 mayinclude a plurality of discrete compartments/chambers (e.g., an array ofwells), each capable of holding mixtures of cells 180, beads 160, andmagnetic stirrers 156 in isolation from one another. In someembodiments, the sample container 120 is pre-packed with a magneticstirrer and/or cell lysis beads, as well as chemicals and/or enzymesthat facilitate cell lysis and preserve the bioactivity of the targetmolecules.

The system 100 may comprise a plurality of sample containers 120, aplurality of filter tips 124, one or more reagent racks 132 or acombination thereof. The sample container rack 108 may be configured tohold multiple sample containers 120 and can be placed on a supportsurface of the housing 104 for simultaneous processing of multiplesamples 144. Likewise, the filter tip rack 112 may be configured to holdmultiple filter tips 124 and can be placed on a support surface of thehousing 104 for simultaneous processing of multiple samples 144. Thesample container rack 108 may also be used as holder of the samples 144for storage purpose. For example, multiple sample containers 120 may beplaced in the sample container rack 108 and stored in a refrigerator orfreezer prior to analysis.

Referring now to FIG. 3 , when in use, the sample containers 120 and thecylindrical magnet 116 are configured such that when the cylindricalmagnet 116 is rotated about its longitudinal axis, the magnetic stirrer156 in the sample container spins and agitates the beads 160 insufficient force to cause disruption and homogenization of cells 180.

The cylindrical magnet 116 may have a number of magnet geometries orconfigurations. In one embodiment, the magnet has magnetic poles (i.e.,north and south) symmetrically disposed along and around thelongitudinal axis of the magnet. The magnet may have a plurality ofopposing poles alternating around and about the longitudinal axis,preferably an even number, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 and 24. In other embodiments, the cylindrical magnet has opposingmagnetic poles disposed at opposite longitudinal ends of the magnet. Inyet other embodiments, the cylindrical magnet is an electromagnet.

The magnet 116 may be rotated above, below or by the side of the samplecontainer 120 about an axis that passes through the center of the magnet116. In certain embodiments, the sample container(s) 120 are placedvertical to the surface on which the sample container(s) 120 reside onand the magnet 116 is rotated about an axis that is also vertical to thesurface on which the sample container(s) 120 reside. In otherembodiments, the sample container(s) 120 are placed vertical to thesurface on which the sample container(s) 120 reside on and the magnet116 is rotated about an axis that is parallel to the surface on whichthe sample container(s) 120 reside. In yet other embodiments, the samplecontainer(s) 120 are placed vertical to the surface on which the samplecontainer(s) 120 reside on and the magnet 116 is rotated about an axisthat forms an angle with the surface on which the sample container(s)120 reside. The angle is greater than 0 degree but smaller than 180degrees.

FIG. 3 shows the relative positions of the magnet 116 relative to asample container 120. The magnet 116 rotates about an axis A and causesa magnetic stirrer 156 in the sample container 120 to rotate in the samedirection along an axis B that is parallel to axis A. While only oneaxis B is shown in FIG. 3 , a person skilled in the art would understandthat the magnetic stirrer 156 may rotate about other B axes that areparallel to other A axes shown in the figure. The rotating magneticstirrer 156 collides with beads 160 and lyses cells 180 in the process.The magnet 116 may be positioned alongside, above, below or diagonallyfrom the sample container 120, which is placed vertical to the surface190 on which the chamber(s) (or the holder of the sample container 120.

The sample container 120, and particularly the sample 144, beads 160,and magnetic stirrer 156, are located within an operational range of avarying magnetic field. For example, the sample container 120 may belocated within an operational range of a rotating magnetic field, e.g.,by placing the container 120 adjacent to or in the proximity of thecylindrical magnet 116. The varying magnetic field drives motion of themagnetic stirrer 156, such as rotational motion, reciprocation, or acombination thereof, among others, which in turn drives motion of thebeads 160, the cells, and the liquid medium. In some embodiments, thesample suspension 144 is stirred with the magnetic stirrer 156 at arotational speed and for durations sufficient to lyse the cells insidethe container 120. The appropriate rotation speed and duration areapplication dependent and can be empirically determined by a person ofordinary skill in the art. Generally speaking, the rotational speedsufficient to lyse the cells, is determined by factors such as the typeof cells, the concentration of sample suspension 144, the volume of thesample suspension, the size and shape of the magnetic stirrer 156, theamount/number, size, shape and hardness of the cell lysis beads 160, andthe size and shape of the sample container 120.

In certain embodiments, the magnetic stirrer 156 is rotating at a speedbetween 1000-6000 rpm, preferably about 5000 rpm, for a time periodbetween 1-600 seconds, preferably about 90-120 seconds. In certainembodiments, a sample container 120 (e.g., in the shape of a urinalysiscup or tube) is placed in a rack on a magnetic stirrer and is stirred atthe highest speed setting (>1000 rpm). In other embodiments, the samplecontainer 120 is a well in a microplate such as an ELISA plate. In otherembodiments, the sample container 120 is a cylinder shaped containerwith a sample inlet and a sample outlet.

In certain embodiments, the speed of rotation of the magnetic stirrer156 is increased to increase lysis efficiency and reduces the timerequired to achieve lysis. In certain other embodiments, the speed ofrotation is regulated so that only certain types of cells are lysed. Forexample, in a sample suspension 144 containing multiple types of cells180, the stirrer 156 may rotate at a first speed to lyse a first set ofcells and then rotate at a second speed to lyse a second set of cells.In other embodiments, the container 120 is coupled to a temperatureregulation module that controls the temperature of the sample suspension144 before, during and/or after the lysing process. In certainembodiments, the temperature of the sample suspension 144 is maintainedat 2°−8° C. In some embodiments, the sample suspension 144 is heated to40°−80° C., 50°−70° C., or about 60° C., before, during and/or after thelysing process (e.g., during the rotation of the magnetic stirrer).

The magnetic stirrer 156 may be made of metal or metal alloy. In oneembodiment, the magnetic stirrer 156 is made of stainless steel. Inother embodiments, the magnetic stirrer 156 is made from an alloy corecoated with a chemically inert material, such as polymer, glass, orceramic (e.g., porcelain). Exemplary alloy core materials includeneodymium iron boron and samarium cobalt. Exemplary coating polymersinclude biocompatible polymers, such as PTFE and parylene.

The magnetic stirrer 156 can be of any shape and should be small enoughto be placed into the sample container 120 and to move or spin or stirwithin the container 120. The magnetic stirrer 156 can be a bar-shaped,cylinder-shaped, cross-shaped, V-shaped, triangular, rectangular, rod ordisc-shaped stirrer 156, among others. In some embodiments, the magneticstirrer 156 has a rectangular shape. In some embodiments, the magneticstirrer 156 has a two-pronged tuning fork shape. In some embodiments,the magnetic stirrer 156 has a V-like shape. In some embodiments, themagnetic stirrer 156 has a trapezoidal shape. In certain embodiments,the longest dimension of the stirrer 156 is slightly smaller than thediameter of the container (e.g., about 75-95% of the diameter of thecontainer).

The cell lysis beads 160 can be any particle-like and/or bead-likestructure that has a hardness greater than the hardness of the cells.The beads 160 may be made of plastic, glass, ceramic, mineral, metaland/or any other suitable materials. In certain embodiments, the beads160 may be made of non-magnetic materials. The beads 160 may berotationally symmetric about at least one axis (e.g., spherical,rounded, oval, elliptic, egg-shaped, and droplet-shaped particles). Incertain embodiments, the beads 160 have polyhedron shapes. In otherembodiments, the beads 160 are irregularly shaped particles. In someembodiments, beads 160 are particles with protrusions. The beads 160 mayhave diameters in the range of 10-1,000 μm, 20-400 μm, or 50-200 μm,among others. The amount of beads 160 added to each lysis container mayrange from about 1-10,000 mg, 1-1000 mg, 1-100 mg, 1-10 mg, amongothers.

After the cells have been lysed, the cell lysate is drawn into asuitable filter tip 124 to allow for the nucleic acids to bind to thefilter matrix 126 therein (see FIG. 4 ). Typically, the lysate is passedthrough the filter matrix 126 at least two times before expelling theunbound portion out the same end of the filter tip 124. At this point,the bound nucleic acids in the filter tip 124 can be stored in a sealedcontainer for further analysis at another time. Alternatively, the boundnucleic acids can be eluted from the filter tip using a suitable elutionbuffer as further described below.

FIG. 4 depicts an exemplary filter tip. The filter tip 124 comprises aporous monolithic binding filter matrix 126 inserted into a pipette tip127. The monolith binding filter matrix 126 comprises a monolithadsorbent or monolithic adsorbent material. The porous monolithicmaterial binds specifically to nucleic acids and is composed of a rigid,self-supporting, substantially monolithic structure. In someembodiments, the porous monolithic material does not include additionalmaterials that provide nucleic acid affinity. In some preferredembodiments, the porous monolithic material is a glass-based monolithicmaterial such as a glass frit. In certain embodiments, the glass frit isa sintered glass frit. The porosity of the porous monolithic material,such as a glass frit or sintered glass frit, is application dependent.In general, the porous monolithic material should have a porosity thatallows for a desired sample flow rate for a particular application andis capable of retaining nucleic acids in a desired size range. In someembodiments, the monolith binding filter matrix 126 is a glass fritconsisting of two sections (126 a and 126 b) with different porosity.

In some embodiments, the porous monolithic material is a glass frit orsintered glass frit having a porosity (i.e., an average pore size) inthe range of 2-400 micron, 2-300 micron, 2-220 micron, 2-200 micron,2-180 micron, 2-160 micron, 2-140 micro, 2-120 micro, 2-100 micron, 2-80micron, 2-60 micron, 2-40 micron, 2-20 micron, 2-16 micron, 2-10 micron,2-5.5 micron, 4-400 micron, 4-300 micron, 4-220 micron, 4-200 micron,4-180 micron, 4-160 micron, 4-140 micro, 4-120 micro, 4-100 micron, 4-80micron, 4-60 micron, 4-40 micron, 4-20 micron, 4-16 micron, 4-10 micron,4-5.5 micron, 10-400 micron, 10-300 micron, 10-220 micron, 10-200micron, 10-180 micron, 10-160 micron, 10-140 micro, 10-120 micro, 10-100micron, 10-80 micron, 10-60 micron, 10-40 micron, 10-20 micron, 10-16micron, 16-400 micron, 16-300 micron, 16-220 micron, 16-200 micron,16-180 micron, 16-160 micron, 16-140 micro, 16-120 micro, 16-100 micron,16-80 micron, 16-60 micron, 16-40 micron, 40-400 micron, 40-300 micron,40-220 micron, 40-200 micron, 40-180 micron, 40-160 micron, 40-140micro, 40-120 micro, 40-100 micron, 40-80 micron, 40-60 micron, 100-400micron, 100-300 micron, 100-220 micron, 100-200 micron, 100-180 micron,100-160 micron, 100-140 micro, 100-120 micro, 160-400 micron, 160-300micron, 160-220 micron, 160-200 micron, 160-180 micron, 200-400 micron,200-300 micron, or 200-220 micron. In other embodiments, the porousmonolithic material is a glass frit or sintered glass frit having twosections (126 a and 126 b) of different porosity. Each section may havea porosity in a range described above (e.g. a 4-10 micron section and a16-40 micron section, or a 16-40 micron section and a 100-160 micronsection).

In some embodiments, the filter has a thickness in the range of 1=30 mm,1-25 mm, 1-20 mm, 1-15 mm, 1-10 mm, 1-8 mm, 1-6 mm, 1-4 mm, 2-30 mm,2-25 mm, 2-20 mm, 2-15 mm, 2-10 mm, 2-8 mm, 2-6 mm, 2-4 mm, 4-30 mm,4-25 mm, 4-20 mm, 4-15 mm, 4-10 mm, 4-8 mm, 4-6 mm, 6-30 mm, 6-25 mm,6-20 mm, 6-15 mm, 6-10 mm, 6-8 mm, 8-30 mm, 8-25 mm, 8-20 mm, 8-15 mm,8-10 mm, 10-30 mm, 10-25 mm, 10-20 mm, 10-15 mm, 15-30 mm, 15-25 mm,15-20 mm, 20-30 mm, 20-25 mm, or 25-30 mm.

In some embodiments, the porous monolithic material may be modified withone or more materials having affinity to the molecules of interest, suchas polynucleotide, protein, lipid or polysaccharide. In someembodiments, the porous monolithic material may be modified with one ormore materials having affinity to nucleic acids.

In some embodiments, the filter is made of a porous glass monolith, aporous glass-ceramic, or porous monolithic polymers. In someembodiments, the porous glass monolith is produced using the sol-gelmethods described in U.S. Pat. Nos. 4,810,674 and 4,765,818, which arehereby incorporated by reference. Porous glass-ceramic may be producedby controlled crystallization of a porous glass monolith. In preferredembodiments, the porous glass monolith, porous glass-ceramic or porousmonolithic polymer is not coated or embedded with any additionalmaterials, such as polynucleotides or antibodies, to improve its bindingaffinity to nucleic acids.

In some preferred embodiments, the filter is made of a finely porousglass frit through which a liquid sample may pass. The porous glass fitis not coated or embedded with any additional materials, such aspolynucleotides or antibodies, to improve its affinity to the nucleicacids or other molecules of interest. Suitable substrates for purifyingnucleic acids include porous glass frits made of sintered glass, whichare formed by crushing beads in a hot press to form a single monolithicstructure. The uniform structure of the frit provides predictable liquidflow inside the frit and allows the eluent to have similar fluiddynamics as the sample flow. The predictable liquid flow allows for highrecovery during the elution process.

Although the filter matrix 126 is typically placed in a pipette tip 127,it may also be fitted into columns, syringes or other housings ofdifferent volumes and shapes. Liquid solutions may be passed through thefilter matrix 126 using various devices, including manual or automaticpipettes, syringes, syringe pumps, hand-held syringes, or other types ofautomated or manual methods for moving liquid across the filter matrix126.

As shown in FIGS. 5A and 5B, the system may further include one or morereagent racks 132 disposed in a housing. The reagent rack 132 isconfigured to hold one or more reagents. The reagent rack 132 may be inthe form of a tray into which reagents can be poured when ready for use.In this case, the reagents may be poured into the tray to facilitatereceipt of the reagents into multiple pipette tips for delivery tomultiple sample wells during the processing of samples 144.Alternatively, the reagent rack 132 may be in the form of a block ormultiwell plate (e.g., 24-well, 96-well etc.) comprising a plurality ofwells 152, whereby each of the plurality of wells is configured to holdany of the reagents for processing a separate sample 144. In certainembodiments, the wells 152 may be prefilled with the reagents and sealedwithin the rack 132. In some embodiments, the rack 132 is located nearthe sample container rack 108 and/or the filter tips rack 112 (FIG. 5B).

The sample purification system 100 may be manually operated or it may beconfigured to be run in a semi-automated or fully automated matter byprogrammable logic. In certain embodiments, the system may furtherinclude an automated pipetting system 136 (FIG. 5A) and one or morerobotic arms (not shown) configured to automatically dispense reagentsfrom one or more reagent racks 132 into a plurality of sample containers120 and dispose of sample materials and used reagents into suitabledisposal receptacles 172 in a predetermined, computer controlled manner(FIG. 5B).

In one mode of operation, reagent racks 132 are in the form of multiwellplates (e.g., 24-well, 96-well etc.). Preferably, the mixtures are mixedby use of automated liquid handling as this will reduce the amount ofwork that needs to be done in order to prepare the mixtures to beinvestigated. Automated sampling protocols may also be performed bymeans of robotics using equipment and methods known in the art.

Any suitable machinery or equipment may be used to move the samples 144through the automated purification system 100 and its various processingsteps. For example, the systems 100 employed herein can use a variety ofrobotics known in the art to automate the movement of samples 144,reagents and other system components. Exemplary robotic systems havecapabilities to move samples on one, two, or three axes and/or to rotatesamples about one, two, or three axes. Exemplary robotics move on atrack which may be situated above, below, or beside a workpiece.Typically a robotic component includes a functional component, e.g., arobotic arm capable of griping and/or moving a workpiece, inserting apipettor, dispensing a reagent, aspirating, etc. A “robotic arm”, asused herein, means a device, preferably controlled by a microprocessor,that physically transfers samples 144, containers 120, filter tips 124,sample container racks 108, filter tips racks 112 and reagent racks 132from one location to another. Each location can be a unit in theautomated purification system 100. Software for the control of roboticarms is generally available from the manufacturer of the arm.

Robotics may be translated on a track, e.g., on the top, bottom, or sideof a work area, and/or may include articulating segments which allow thearm to reach different locations in the work area. Robotics may bedriven by motors known in the art, which may be, for exampleelectrically, pneumatically, or hydraulically powered. Any suitabledrive control system may be used to control the robotics, such asstandard PLC programming or other methods known in the art. Optionallythe robotics include positional feedback systems that optically ormechanically measure position and/or force, and allow the robot to beguided to a desired location. Optionally, robotics also include positionassurance mechanisms, such as mechanical stops, optical markers or laserguides, that allow particular positions to be repeatedly obtained.

Exemplary automated sampling protocols may utilize, for example, anEppendorf epMotion 5070, epMotion 5075, Hamilton STARlet, STAR andSTARplus liquid handling robots. Such protocols may be adapted for RNAisolation, genomic DNA isolation from whole blood, tissues, saliva,swabs, as well as circulating cell-free DNA such as circulating tumorDNA and circulating fetal DNA extraction and enrichment from maternalplasma.

Methods for Purifying Nucleic Acids

In another aspect, a method for purifying nucleic acids from a sample,includes the steps of (a) providing a nucleic acid purification systemin accordance with the present disclosure; (b) introducing into a samplecontainer the sample, a magnetic stirrer and a plurality of beads; (c)rotating the cylindrical magnet about its longitudinal axis so that themagnetic stirrer spins and agitates the beads to undergo chaotic mixingof cellular contents to a degree sufficient for homogenizing the sampleand disrupting the cells in the sample to form a cell lysate; (d)flowing at least a portion of the cell lysate through a first opening ofa filter tip so that nucleic acids in the cell lysate bind to the filterin the filter tip; (e) expelling an unbound portion of the cell lysatefrom the filter tip via the first opening, where the unbound portionpasses through the filter at least two times before exiting the filtertip; and (f) eluting the nucleic acids bound to the filter by flowing anelution buffer in through the first opening of the filter tip andexpelling the elution buffer from the filter tip via the first opening,wherein the elution buffer passes through the filter at least two timesbefore exiting the filter tip.

Any mode of performing the method according to the present applicationcan be employed, including fully manual, semi-automated or fullyautomated protocols. However, the attributes, adaptability, simplicityand workflow of the filter tip allow for it to be readily adapted,automated, and effective for a number of clinical sample matrices, inputsample volumes, and liquid handling systems. Thus, in a preferredembodiment, the mode of operation includes some kind of automation. Inone embodiment, the method for purifying nucleic acids comprises anautomated pipetting system and one more robotic arms configured toautomatically dispense reagents into one or more sample containers anddispose of sample materials and reagents into suitable disposablereceptacles in a predetermined manner. In this case, each of theabove-described steps are repeated in each of a plurality of samplecontainers using an equivalent number of filter tips in combination withone or more reagent racks.

Samples suspected of containing MTB present a potential risk to theuser. Accordingly, the sample may be pre-treated by heating and/orinclusion of reagents suitable for inactivating microbes present in thesample to mitigate this risk. Inactivation of microbes, such as MTB, maybe carried out by heating (e.g., 90° C., 5 min.) to denature activeproteins, enzymatic digestion of cell wall structures, mechanicaldisruption to physically disrupt or inactivate the cells, chemicaltreatment or a combination thereof.

Chemical inactivation offers the potential to reduce or eliminate theneed for heat. When processing a sample for culturing, simple reagentsare used to digest the sputum and disinfect the sample. For culturing,it is important to inactivate other flora present in the sputum sample,so that MTB can grow without being overtaken by other faster-growingbacteria. The decontamination or inactivation step, which may usereagents such as sodium hydroxide (e.g., 3-5%) or cetylpyridiniumchloride, is preferably selected to inactivate all other bacteria, butkeep MTB cells with a thicker, more robust cell wall, intact and alive.However, depending on the method used, 20 to 90% of the MTB cells can bekilled during this process. With regard to nucleic acid purification,however, the MTB cells do not need to be alive or intact, as long as thebacterial genomic DNA is still able to be amplified.

Inactivating reagents are preferably chosen to allow for limiteddilution of the sample and/or low pH for compatibility to silicabinding. These reagents may be added to the sample at the time ofcollection. In some embodiments, hydrogen peroxide, alcohols, such asethanol and o-phenylphenol (e.g., 0.2-0.5%) may be used as primaryactive ingredients. Hydrogen peroxide (H₂O₂) may be used as a chemicalsterilant from 6-25% concentrations and is very stable in solution. Whenmixed with 0.85% phosphoric acid, H₂O₂ is active at low pH. Ethanolalone (e.g., at 95%) can inactivate MTB in sputum or water in 15seconds. 0-phenylphenol, an agricultural fungicide, is used at 0.1-0.41%with either ethanol or isopropanol in PHENO-CEN, SRAYPAK, andCLIPPERCIDE spray disinfectants. In addition, 0-phenylphenol may be usedat low reagent to sample ratios at room temperature in 15 min and it maybe used in combination with ethanol or isopropanol or with 6.65%2-Benzyl-4-chlorophenol in Low pH Phenolic 256 (50%-100%)

The volume ratio of inactivating reagents to sample volume willtypically range from about 0.1:1 to 3:1.

Inactivation of microbes in the primary specimen container is importantfor providing a BSL-1 compatible workflow (i.e., the workflow does notrequire a biosafety cabinet). Many protocols involve sample transferprior to disinfection, a practice which can produce aerosols, and infectthe user. Accordingly, BSL-1 compatibility requires careful attention tosample transfers prior to disinfection, particularly those that canproduce aerosols and infect the user.

Depending on the nature of the sample, the sample may be initiallyliquefied to reduce its viscosity and heterogeneity for consistentsample processing. Sputum samples present a particular challenge. MTB insputum is one of the most challenging cell and sample types to processdue to the lipid-rich hydrophobic cell wall of acid fast bacilli and theviscous, heterogeneous nature of sputum. Standard extraction methods forsputum typically start with a process of sedimentation, which ofteninvolves the treatment with N-acetyl-L-cysteine (NALC) and sodiumhydroxide (NaOH) followed by centrifugation, decanting, andre-suspension. Accordingly, when processing highly viscous samples, suchas sputum, the sample may be subjected to chemical treatment in order toreduce the viscosity so that subsequent processing steps (e.g., MagVor)are not impeded. Exemplary mucolytic reagents for addition to the sampleinclude, but are not limited to NALC, zephiran-trisodium phosphate(Z-TSP), benzalkonium, and Primestore™. (Longhorn Vaccines &Diagnostics, San Antonio Tex.), which contains a special formulation tolyse bacteria and stabilize RNA and DNA. In one embodiment, liquefactionof samples by chemical treatment with mucolytic agents is carried outfor 20 minutes at 60° C. Patient sputum sample may be typicallycollected in volumes between 1-10 ml, 5-10 ml or greater.

Sputum has a viscosity range from about 100-6,000 cP (mPa's) with ashear rate at 90 s⁻¹. The viscosity, measured in mPas, is determined byshear strength divided by shear rate. Preferably, the sample isliquefied to reduce the viscosity of sputum by at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%.

When processing a sample, at least one magnetic stirrer and a pluralityof cell lysis beads are present in the sample container. A user maysimply add a sample suspension into the sample container, place thesample container adjacent to the cylindrical magnet, and stir the samplesuspension by rotating the magnet at a speed sufficient for the rotatingmagnetic field to cause rotation of the magnetic stirrer and stirring ofthe cell lysis beads in the sample container in a manner sufficient tohomogenize and lyse the cells.

The sample suspension, cell lysis beads and the magnetic stirrer may beplaced into the sample container in any order. In some embodiments, thesample suspension is added to the sample container before the cell lysisbeads and the magnetic stirrer. In other embodiments, the cell lysisbeads and/or the magnetic stirrer are placed into the sample containerbefore collection of the sample.

In certain embodiments, lysing of particular cell types can befacilitated by adding additives to the sample suspension prior to and/orduring the stirring step. Examples of additives include enzymes,detergents, surfactants and other chemicals such as bases and acids. Ithas been found that alkaline conditions (e.g., 10 mM NaOH) may enhancethe lysis efficiency during stirring for certain types of cells. Thesample suspension may also or alternatively be heated during stirring toenhance the lysis efficiency. Additives, however, can be detrimental todownstream processing steps including nucleic acid amplification anddetection and should be eliminated when possible to simplify theprocess.

The stirrer/beads combination provides many advantages over conventionallysing methods. The stirrer/beads method is much faster than chemicaland enzymatic approaches, and provides improved cell or virus lysis overmany other types of physical lysis methods. The stirrer/beads method isalso amenable to automation using robotics and/or microfluidics. Thecylindrical magnet is reusable, doesn't require precise alignment withthe container, and can drive a plurality of chambers. The magneticstirrer is low-cost, enabling its use in a single-use disposable.

Following the MagVor step, a suitable binding buffer containing one ormore chaotropic agents are added to the sample container to facilitatebinding of nucleic acids to the filter matrix 126. BOOM chemistry, orchaotropic binding of nucleic acids to silica, is most efficient whenthe solution pH is less than 7. In this case, high ionic strengthsolutions containing lithium or sodium chloride, or guanidine-based ionsare typically combined with an aliphatic alcohol, such as ethanol orisopropanol to “salt out” the DNA and promote nucleic acid binding,respectively. A suitable binding buffer is used at a concentration sothat when it is added to the processed sample, the resultant volume iswithin the range of the volumetric capacity of the filter tip. Thisreduces the number of aspirate and dispense cycles, and thus, the totalprocessing time.

In certain embodiments, the binding buffer is added to the sample andincubated for 10 minutes at 60° C. following MagVor. In otherembodiments, chaotropic agents and aliphatic alcohols are included inthe liquefaction step, prior to the MagVor step. In other embodiments,the inactivation, homogenization and lysis steps are carried out in asingle step in as little as 15 minutes.

Exemplary chaotropic agents include, but are not limited to chaotropicsalts, such as guanidinium thiocyanate, guanidine isothiocyanate,guanidine hydrochloride, guanidinium chloride urea, thiourea, sodiumdodecyl sulfate (SDS), cetylpyridinium chloride, sodium chloride,lithium chloride, potassium chloride, sodium perchlorate, lithiumperchlorate, sodium iodide, and potassium iodide; aliphatic alcohols,such as butanol, ethanol, propanol and isopropanol; phenol and otherphenolic compounds.

In some embodiments, to promote selective binding of high molecularweight (HMW) nucleic acids to the filter matrix, an aliphatic alcohol,such as isopropanol is provided in a range between about 0% to about10%, preferably between about 4% to about 6% (optimal=4.7%) and achaotropic salt, such as guanidine isothiocyanate and/or guanidinehydrochloride is provided in a range between 1.0 M to 4.0 M, preferablybetween about 3.0 M to about 4.0 M.

In some embodiments, to promote binding (and concentration) of lowmolecular weight (LMW) nucleic acids to the filter matrix, an aliphaticalcohol, such as isopropanol is provided in a range between about 10% toabout 25%, preferably between about 15% to about 20% (optimal=17.7%).

In other embodiments, to promote isolation of MTB DNA from sputum, analiphatic alcohol, such as ethanol may be provided in a range betweenabout 20% to about 60%, preferably between about 30% to about 50%(optimal=44%)

Following the above-described inactivation and homogenization steps, thepH of the solution should be adjusted, as necessary, to achieve a pHbelow 7. Where the pH is above 7, the solution can be neutralized with amild acid, such as potassium acetate or sodium phosphate. This will benecessary when using NaOH or O-phenylphenol, which have a pH>11. Low pHphenolic and hydrogen peroxide reagents are inherently acidic and willmost likely not need an additional buffer.

In one embodiment, binding of nucleic acids to the filter matrix may becarried out by attaching a filter tip 124 to a syringe 176 via luer lockconnections between the two. In another embodiment, the filter matrix isin the syringe 176. An exemplary filter tip 124 is shown in FIG. 4 . Thefilter tip 124 comprises a porous silica matrix 126 embedded inside of atip body 127, an aerosol filter 128 to prevent contamination andexposure to the user, and a tip cap 129 to help maintain a closedsystem. In one embodiment, the cap 129 is connected to the filter tip124 with an insert. In some embodiments, the cap is an ordinary Falcontube cap 208. The filter tip 124 is configured to allow a liquid sampleto flow through the matrix with each aspiration and dispense cycle ofthe filter tip 124. Using a syringe 176 or other suitable device, thecell lysate in the container 120 is passed up through the distal end ofthe filter tip 124 so that nucleic acids in the cell lysate bind to thefilter matrix 126 in the pipette tip 127. Typically, the cell lysate isdrawn up and down through the filter matrix 126 so that the lysate andunbound portion passes through the filter matrix 126 at least two timesbefore expelling the unbound lysate fraction out through the distal endof the pipette tip 127 into a suitable disposal receptacle 172.

Combining the sample with chaotropic reagents dehydrates the DNA andsilica to promote adsorption to the porous silica matrix 126. Subsequentaspiration and dispense cycles (2-3×) of a wash buffer remove impuritiesfrom the matrix 126. At this point, filter tip 124 containing nucleicacids bound to the filter matrix 126 can be stored in a sealed container120 for further analysis at another time. Alternatively, the boundnucleic acids can be eluted from the filter tip using a suitable elutionbuffer as further described below.

Nucleic acids have been shown to be very stable on solid supports,including silica, particularly when stored under dehydrated conditionswithout any additional stabilizers. Accordingly, in another aspect, thepresent application provides a means for stabilizing the purifiednucleic acids for transport in the form of a single-use disposabletransport device 200 comprising a luer lock adapter 204 attached to thetop side of the cap 208 of a suitable holding tube 212 (e.g., 50 mLconical tube), such that the filter tip 124 attaches to the bottom sideof the tube cap 208 (FIG. 6 ).

Before use (i.e., elution of the nucleic acids for analysis), the filtertip 124 attached to the cap 208 is removed from the holding tube 212 andattached to a syringe 176 as shown in FIG. 2 . In this case, the usercan readily insert and remove the filter tip 124 via the luer lockadaptor 204 while holding onto the tube cap 208, such that the holdingtube 212 shields the user and the filter tip 124 from contamination.When this sequence is completed, the porous silica filter matrix 126with bound nucleic acids can be dried and the capped filter tip 124 isscrewed onto the empty holding tube 212 for transport. Duringtransportation, the holding tube 212 protects the filter tip 124 fromcontamination. The stabilized nucleic acids can be rehydrated later withelution buffer and eluted off into a storage tube for long-term frozenstorage or eluted directly into a detection assay device or sample tubeusing a similar automated system as used at the clinic or simply byusing a disposable syringe 176. In the latter case, the syringe 176 canserve as the mechanism for the aspiration and dispense cycles of theelution buffer across the silica matrix 126.

In preparation for molecular analysis, aspiration and dispense cycles(2-3×) of an elution buffer remove the bound nucleic acids from thematrix 126. Completion of the process results in purified nucleic acidsin a PCR-compatible buffer. This approach allows for flexibility withrespect to format such that it can be used with a liquid handling systemfor high throughput applications or with a simple pipette tip for lowthroughput applications.

FIG. 7 illustrates an exemplary sequence of steps for MagVor/filter tippurification of nucleic acids from sputum. A sputum sample suspected ofcontaining MTB is collected with a sample container. A chemical reagentmix, including inactivating reagents and mucolytic agents are added(Step 1). The sample container is then placed on an extraction stand (orrack) and the sample contents are subjected to magnetically-inducedvortexing (MagVor) for 2-15 minutes, preferably about 10 minutes (Step2). Following lysis of the cells, the beads are allowed to settle forabout 1-2 minutes and binding buffer is added to the container (Step 3).The user attaches a filter tip to the bottom side of the tube cap/luerlock adaptor in FIG. 6 and connects a syringe to the top side of thetube cap/luer lock adaptor using the luer lock connection (Step 4). Theuser pierces the filter tip through the cap over and into the container,and draws the cell lysate into the tip and moves the syringe lever upand down past the filter matrix 2-3 times to facilitate binding to thefilter matrix, whereby the unbound portion is passed back into the tube(Step 4). Following this binding step, the sample container is replacedwith a fresh tube containing wash reagent, and the filter matrix iswashed with wash buffer, which is collected in the tube (Step 5).Following the wash step, the tube is raised out of the liquid. In someembodiments, the filter tip is further dried by dispensing air throughthe filter matrix (Step 6). In some embodiments, several rounds of airdrying are performed to reduce residual wash reagent. Then the userdetaches the filter tip/cap adaptor from the syringe, places the filtertip back in a fresh holding tube containing dessicant, and connects thefilter tip/cap adaptor to the holding tube for storage (Step 7). Thenucleic acids in the filter tip are stable for transport or can elutethe purified nucleic acids from the filter tip for PCR analysis etc.Elution of the nucleic acids may be carried out by passing elutionbuffer through the filter matrix 2-3 times before collection.

Additives, such as trehalose, 0.1% Triton-X-100 or DNAstable® Plusreagent (Biomatrica) may be included with the elution buffer or added tothe eluted nucleic acids to enhance their stability.

In certain embodiments, the method further includes the steps of elutingthe nucleic acids, amplifying the eluted nucleic acids with primersspecific for a predetermined target, and determining whether the samplecontains nucleic acids corresponding to the target. Preferred targetsfor detection include bacterial and viral pathogens found in sputum,including/but are not limited to, MTB, Staphylococcus aureus,methicillin resistant Staphylococcus aureus (MRSA), Streptococcuspyogenes, Streptococcus pneumoniae, Streptococcus agalactiae,Haemophilus influenzae, Haemophilus parainfuluezae, Moraxellacatarrhalis, Klebsiella pneumoniae, Escherichia coli, Pseudomonasaeruginosa, Acinetobacter sp Bordetella pertussis, Neisseriameningitidis, Bacillus anthracis, Nocardia sp., Actinomyces sp.,Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella species,Pneumocystis jiroveci, influenza A virus, cytomegalovirus, andrhinovirus.

The system 100 described herein can detect MTB at levels of less than1,000 cells/ml, preferably less than 100 cells/ml, more preferably lessthan 50 cells/ml, most preferably less than 10 cells/ml. Given that 1colony forming unit (cfu) is roughly equivalent to 10 cells, the abovesystem can be used to provide a detection of at least 100 cfu/ml, 10cfu/ml, 5 cfu/ml or even 1 cfu/ml.

It should be recognized, however, that every clinical sample is unique,and will vary one to the next in viscosity, particulates, mucus, surfacecontaminants, microbial and/or human genetic backgrounds. Given expectedvariations in clinical sample composition and intended uses of anautomated filter tip sample preparation protocol, it may therefore benecessary to modify certain steps in a filter tip procedure in order toachieve desired results.

For example, while the filter tips described herein have a relativelylarge pore size, sample homogenization and liquefaction is veryimportant for efficient cell lysis, and subsequent binding steps to thefilter matrix. With homogenous and well-liquefied lysates, samples canalso be passed over the filter tip with higher flow rates, which reducesthe overall sample processing time. As demonstrated with thelarge-volume plasma protocol below, large input sample volumes can beeffectively processed with a filter tip, which provides users theopportunity to thoroughly homogenize and liquefy difficult samples(on-line or off-line), with only minor concern over input samplevolumes.

Further, it should be appreciated that slower flow rates during nucleicacid binding or elution typically result in higher nucleic acid yields,albeit at the expense of total processing time. Slower flow rates willalso minimize the extent of DNA shearing.

Complete drying of the filter matrix is recommended to prevent residualorganic solvents from co-eluting with the purified nucleic acid sampleand inhibiting downstream processes or tests. Because the filter tip isnot dried via centrifugation or vacuum filtration, it is important tomaximize both the flow rate and cycle numbers during the drying step.

Because the geometry, filter tip material, and attachment method to therobotic channel arms are unique for each instrument manufacturer, adifferent filter tip construct is required for each liquid handlingsystem. The filter matrix dimensions (diameter, thickness, and poresize) do correlate with nucleic acid binding capacity (and elutionefficiencies), as is expected for any solid-phase extraction technique.While thick (>4 mm) matrices may be embedded into a 1 ml filter tip toincrease nucleic acid binding capacity for large-volume samples and/orequalize the matrix binding capacity across specific filter tip formats,there is a tradeoff between filter tip thickness and flow rates duringthe initial binding step (in the presence of crude lysates). Thus, it issometimes advantageous to embed larger-diameter matrices intolarger-volume filter tips for the initial steps of an automatedprotocol, (e.g., the 5 ml Hamilton/Akonni TruTips® for large-volumeextractions). Given the specific filter tip configurations dictated bythe manufacturers of liquid handling robots, however, it is notreasonable to expect the filter tip nucleic acid yields to be identicalacross liquid handling platforms from different manufacturers, or acrossdifferent filter tip sizes. Clinical evaluation of automated filter tipprotocols and direct comparisons against commercially-availableautomated systems will be reported in detail elsewhere.

The MagVor/filter tip process has a number of advantages compared withconventional methods. First, the process is compatible with automation.Secondly, confinement of the filter matrix within the pipette tipreduces its susceptibility to cross-contamination. Fibrous silicamatrices on spin disks, on the other hand, can easily rupture andrelease fine particles that can be a source of contamination. Similarly,techniques that rely on the mobility of magnetic beads for purificationintroduces a similar risk. The relatively large porosity of the filtermatrix allows high viscosity samples to flow through the matrix withoutclogging. The multiple aspiration and dispense cycles allow forincreased binding of target nucleic acid when compared withcentrifugation methods employing single passage of samples through amatrix. Moreover, the chaotropic chemistry provides an establishedmethod for removing inhibitors and nucleases, and lends itself tolong-term stability.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

EXAMPLES Example 1: MagVor Homogenization and Lysis

The efficacy of the MagVor system was tested against Bacillusthuringiensis spores, Streptococcus pyogenes, and MTB in raw sputum,using a 1:1 v:v ratio of glass beads to sample volume, 1 mL total samplevolume, and 30 to 120 sec of MagVor lysis. MTB DNA extraction from rawsputum is particularly challenging in view of its mycobacterial cellwall and low (10 bacilli) infectious dose. Lysis efficacy was estimatedby quantitative, real-time PCR of equivalent raw sputum samples beforeand after MagVor treatment. MagVor processing was found to improvenucleic acid detection by an average of 2.5 cycles (nearly 1 log)relative to untreated samples (i.e., aliquots of the same sputum samplebut not processed by the MagVor system).

To investigate the extent to which physical lysis kit components(particles, stir disk, coatings) may interfere with nucleic acidextraction and purification, and analysis of four bead types and threemagnetic disks was undertaken to identify a lysis bead and magnetic diskthat did not generate ultra-fine particulates in solution after MagVorlysis. NPA samples were pooled, confirmed negative for target DNA ofinterest by real-time PCR, and then spiked with intact methicillin MRSAor MTB target cells. Specimens (0.5 mL) were processed in duplicate andlysed with a 10 min MagVor treatment at 5000 rpm. The nucleic acids werepurified with a manual MagVor/filter tip procedure employing aguanidinium-based binding buffer and then analyzed by quantitative,real-time PCR (or RT-PCR). Glass beads readily settled to the bottom ofthe lysis tubes and showed no obvious inhibition or degradation of DNAor RNA during these tests.

As shown in Table 1, a consistent improvement in DNA recovery wasobtained with MagVor treatment compared to no treatment, especially athigher titers.

TABLE 1 Combined MagVor/filter tip recovery of MRSA and MTB DNA fromspiked NPAs Titer MagVor No Treatment Organism (cfu mL⁻¹) Ave. C₁ Ave.C₁ ΔC₁ MRSA 10⁸ 23.69 28.86 5.17 10⁷ 25.50 28.56 3.06 10⁶ 29.32 31.672.36 10⁵ 31.81 33.98 2.17 MTB 10⁷ 17.31 20.38 3.08 H37Ra 10⁶ 21.33 23.822.49 10⁵ 24.33 26.50 2.17 10⁴ 28.79 29.77 0.98 10³ 31.59 32.76 1.17 10²33.63 34.52 0.89

Example 3: Comparison of Integrated MagVor/Filter Tip Prototype toQiagen Nucleic Acid Purification Kits

The nucleic acid lysis and purification efficacy of the integratedsystem was evaluated in comparison to comparable Qiagen nucleic acidpurification kits. Model sample types included MRSA in NPA, influenza Ain NPS, human genomic DNA from whole blood, and MTB in NPA. The Qiagenkits included the DNA Mini Kit (no mechanical lysis, but with 10 minproteinase K treatment), Viral RNA Mini Kit (no mechanical lysis, butwith RNA carrier), and Mini Blood Kit (10 min proteinase K incubation).Since Qiagen does not have a specific kit for MTB extractions, a BDGeneOhm lysis kit was employed in conjunction with a Qiagen Mini DNAextraction kit. BD lysis and Qiagen kits also have a limited inputsample volume, so NPA and NPS samples were processed in 200 μL volumes,and whole blood was processed in 100, 10, and 1 μL volumes. Replicatereagent plates were prepared, sealed, and processed for each sample typeand titer (n=24 extractions per sample), purified nucleic acid analyzedby quantitative, real-time PCR, and average cycle-threshold (Ct) valuescompared to those obtained from comparable Qiagen extractions (n=8extractions per sample). Positive and negative controls were run witheach plate to test for potential cross-contamination.

The results from this analysis are summarized in Table 2, anddemonstrate comparable performance and efficacy relative to the Qiagenkits. Limits of detection for MRSA in NPA and influenza A in NPS wereapproximately 10³ cells or virions/ml for both systems. Human DNA wasreadily recovered from 1 μL of whole blood, and no template controlsshowed no evidence of nucleic acid cross-contamination. These datademonstrate the extensibility and efficacy of the integrated samplepreparation prototype relative to commercially-available, high-qualitysample preparation kits.

TABLE 2 Nucleic acid recovery from MagVor-filter tip system (n = 24)relative to other DNA extraction kit Titer Integrated (cfu mL⁻¹,Prototype Qiagen or μL) (Ave C₁ StDev) (Ave C₁ StDev) MRSA in 10⁶ 29.070.49 30.73 0.12 NPA 10⁵ 33.82 0.87 33.69 0.26 10⁴ 35.74 0.62 36.33 0.68*10³  38.82 0.77 38.86 1.08 NTC Not Detected 10⁶ 23.47 0.30 24.91 0.43Influenza A 10⁵ 27.71 .040 29.45 0.61 In NPS 10⁴ 34.02 0.80 34.61 0.74*10³  37.94 1.14 38.44 0.81 NTC Not Detected Human 100 μL 26.87 0.5926.26 0.21 DNA in  10 μL 29.66 0.21 29.35 0.13 Whole  1 μL 32.14 0.3732.75 0.47 Blood NTC Not Detected MTB in NPA 10⁶ 21.20 0.27 22.04 0.3810⁵ 25.74 0.34 27.78 1.91 10⁴ 28.81 0.46 30.67 0.67 10³ 31.31 0.72 32.820.35 10² 34.35 0.58 35.67 0.32 NTC Not Detected

Example 4: Sputum Liquefaction

Since most specimens submitted for mycobacterial culture arecontaminated with a variety of organisms that can rapidly outgrow themycobacteria, before analysis, respiratory specimens are typicallysubmitted to digestion-decontamination pretreatment. Thus, mycobacteriaare recovered optimally from clinical specimens through use ofprocedures that reduce or eliminate contaminating bacteria whilereleasing mycobacteria trapped in mucin and cells.

NALC-NaOH sedimentation has become the standard for decontaminating anddigesting sputa specimens of non-tuberculous mycobacteria (NTMs).However, the extraction of MTB DNA from raw sputum is particularlychallenging because of the high viscosity and heterogeneity of sputum,on the on hand, an the difficult-to-disrupt cell walls of MTB, on theother hand. Transport of DNA extracts reduces the logistical complexity(in terms of cold transport) compared to raw sputum. While NALC-NaOH isindeed a digestion procedure, NALC rapidly loses its activity, requiringfresh reagents to be reconstituted daily. In addition, the procedurerequires centrifugation, which adds further complexity and equipment.Furthermore, NaOH exposure causes MTB cell death and degrades DNA.

Accordingly, it was of interest to develop a liquefaction method thatcould serve as an alternative for users who have interest in processingraw sputum. Since raw sputum is a difficult specimen type to transfer tosample containers, a single-step sputum liquefaction procedure wasdeveloped using an enzyme solution. By adding 1 part liquid enzyme to 10parts raw sputum and incubating for 15-20 min at 56° C. even highlyheterogeneous and viscous sputum specimens were liquefied to a viscositycomparable with 5-10% glycerol. These liquefied sputum specimens werereadily pipetted and processed with a manual MagVor and filter tipprotocol without causing the magnetic disk in the lysis tube to stoprotating or clogging the filter tip.

Example 5: Extraction of MTB DNA from Raw Sputum

An automated 8-channel prototype system depicted in FIGS. 5A and 5B wasused to demonstrate the feasibility of DNA extraction from raw,TB-positive sputum. Using a Truant TB Fluorescent Stain, de-identifiedpatient specimens were determined by smear microscopy to be either Smear2+ or 4+. Four aliquots of Smear 2+ raw sputum specimens and fouraliquots of Smear 4+ raw sputum specimens were processed according tothe liquefaction protocol in Example 4 and then added to the MagVortubes. The Integrated MagVor/filter tip protocol was then performed byautomated extraction/purification. The eluent was analyzed with a IS6110qPCR assay, and the concentrations of the extracts were found to be3.6±0.7 pg/μL for the Smear 2+ and 49±8 pg/μL for the Smear 4+. Thisdata supports the feasibility of the automated nucleic acid isolationinstrument for extracting DNA from TB positive specimens.

Table 3 shows the results of a dilution series study comparing real-timedetection of MTB using an automated vs. manual MagVor/filter tip system.MTB cells were spiked into 500 μl of TB-negative sputum and sediment(NALC-NaOH processed sputa), where 10 cells is roughly equivalent to 1cfu/ml. Corresponding cell levels corresponding to acid-fast bacilli(AFB) smear positive and AFB smear negative are included for comparativepurposes.

TABLE 3 3 Dilution series study showing real-time detection of MTB DNAby manual and automated MagVor/filter tip systems Average C₁ ± StDev (n= 6 extractions, two replicate PCR_(s per extraction)) Cells NALC-NaOHmL⁻¹ Raw Sputum Sediment Automated 10⁶ 21.24 ± 0.20 21.61 ± 0.12 AFBTruTip 10⁵ 26.24 ± 0.14 25.45 ± 0.41 Smear+ 10⁴ 29.05 ± 0.17 29.12 ±0.32 10³ 31.71 ± 0.39 29.85 ± 0.19 AFB 10² 31.77 ± 0.38 31.19 ± 0.20Smear− 10¹ Inconsistent Inconsistent  0 ND ND Manual 10⁶ 24.98 ± 0.6724.01 ± 0.40 AFB TruTip 10⁵ 26.15 ± 0.54 26.78 ± 0.41 Smear+ 10⁴ 28.18 ±0.56 28.18 ± 0.43 10³ 30.90 ± 0.24 32.14 ± 0.12 AFB 10² 34.07 ± 0.5333.24 ± 0.41 Smear− 10¹ Inconsistent Inconsistent  0 ND ND

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A method for purifying molecules of interest froma sample with a sample purification system that comprises: a housing; anelectric motor disposed in the housing; a sample container rack disposedin the housing, wherein the sample container rack is configured to holdone or more sample containers, a filter device holder disposed in thehousing, wherein the filter holder is configured to hold one or morefilter devices, wherein each of the filter devices comprises a filtermatrix for binding molecules of interest; and a cylindrical magnetadjacent to the side of the sample container rack and external to thesample container rack, wherein the magnet rotates around a central,longitudinal axis of the magnet by an electric motor disposed in thehousing and is located above the bottom of the sample container rack,wherein the method comprises following steps placing a sample containercontaining a liquid sample suspension, a magnetic stirrer and cell lysisbeads on the sample container rack; homogenizing the liquid samplesuspension in the sample container by rotating the cylindrical magnet ata speed such that cells in the sample suspension in the sample containerare lysed by the magnetic stirrer and cell lysis beads; flowing thehomogenized liquid sample suspension through the filter matrix of afilter device from the one or more filter devices under conditions suchthat the molecules of interest bind to the filter matrix, wherein thefilter device is mounted on the filter device holder; washing thefilters matrix of the filter device and eluting the molecules ofinterest bound to the filter matrix from the filter device, therebypurifying the molecules of interest from the sample.
 2. The method ofclaim 1, wherein the sample container is pre-packed with one or moreitems selected from the group consisting of the magnetic stirrers, thecell lysis beads, reagents that facilitate cell lysis and reagents thatpreserve the integrity of the molecules of interest.
 3. The method ofclaim 1, wherein the molecules of interest are nucleic acids.
 4. Themethod of claim 3, wherein the liquid sample suspension comprisessputum.
 5. The method of claim 4, wherein the molecules of interest arenucleic acids and further comprising the step of amplifying themolecules of interest eluted from the filter device with primersspecific for Mycobacterium tuberculosis and determining whether themolecules of interest comprise Mycobacterium tuberculosis DNA.
 6. Themethod of claim 1, wherein the cylindrical magnet has two magnetic polessymmetrically disposed along the longitudinal axis of the magnet.
 7. Themethod of claim 1, wherein the cylindrical magnet has opposing magneticpoles disposed at two opposite ends of the magnet.
 8. The method ofclaim 1, wherein the cell lysis beads are silica beads having diameterswithin a range from 10 μm to 1000 μm.
 9. The method of claim 1, whereinthe magnetic stirrer comprises an alloy core coated with a polymer. 10.The method of claim 9, wherein the alloy core of the magnetic stirrercomprises neodymium iron boron or samarium cobalt and wherein thepolymer is polytetrafluoroethylene (PTFE) car parylene.